Screening And Metabonomics Analysis Of Avermectin High-yieding Streptomyces Avermitilis Mutant

Avermectin,a series of biological pesticides safe to humans and animals,has been widely used in agricultural production.Although the whole genome sequence of avermectin producer Streptomyces avermitilis has been accomplished,the the mechanism of high-yielding of avermectin is uncertain.Therefore research on metabolomics of S.avermitilis is particularly important.Currently,S.avermitilis is the best avermectin production strain.Mutagenesis screening is the most common and effective way to acquire excellent production strain.In this dissertation,atmospheric room temperature plasma(ARTP)technology was employed in the mutation of S.avermitilis.Furthermore,the streptomycin and kanamycin resistance screening combined with 96 deep-well plates for high-throughput screening was used for higher-yielding mutant screening.A stable avermectin high-producing mutant S-2C3 was screened out from 73 streptomycin-resistant mutants,and K-1A6 from 52 kanamycin resistance strains,their avermectin production respectively reached 4.06 mg/mL(18.8%enhancement)and 4.22 mg/mL(23.4%enhancement)as compared to the parent strain 9-39.Metabolomics is the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.In this dissertation,the key steps of S.avermitilis metabolomics sample preparation were investigated,including cell separation,washing,quenching,disruption and extraction conditions.Furthermore,the method for S.avermitilis metabolomics was estabolished.The optimal sample preparation conditions were as follows:separating cells by centrifugation,washing 3 times,quenching with liquid nitrogen,cell disruption with bead mill(2 cycles for 48 s each),and extraction of metabolites(chloroform:ethanol:water=2:2:1).By using this optimized protocol,59 differential compounds have been identified and quantified,including amino acids,organic acids,fatty acids and carbohydrate,etc.The metabolomics method was used to study the metabolic profiling of mutant K-1A6.It turned out that the difference between K-1A6 and starting strain was the avermectin synthetic precursor and oxygen supply.As the synthetic precursor of avermectin,the intracellular level of oxalic acid in K-1A6 was higher than that in 9-39.It is inferred that the sufficient supply of avermectin synthetic precursor,energy and oxygen may be the reason of high production in the mutant strain.In addition,intracellular levels of fatty acids were significantly reduced in K-1A6 as compared to those of 9-39 in the fermentation process,which led to the less consumption of acyl-CoA,the precursor of avermectin,it may be the another reason of the high production of K-1A6.S-adenosylmethionine(SAM)is well known as an important physiological active material.SAM is not only as a methyl donor and involved in the transfer of sulfenyl and ribosomal group,but also participating in regulation of microbial metabolism as signaling molecule.In this dissertation,the effect of SAM on avermectin production of S.avermitilis fermentation was studied,and the metabolomics method was used to find the impact of SAM on the S.avermitilis metabolism characteristics.The results demonstrated that SAM improves the synthesis of threonine,propionic acid and acyl-CoA,which are the precursors of avermectin biosynthesis,so that there are enough precursors for the biosynthesis of avermectin.